Excess glutamate is neurotoxic and contributes to a number of degenerative diseases of the central and peripheral nervous system. One source of glutamate in these tissues is N-acetyl-aspartyl-glutamate (NAAG), a dipeptide found in the brain and peripheral nerves. NAAG hydrolysis is catalyzed by glutamate carboxylase enzymes, including glutamate carboxypeptidase II and glutamate carboxylase III. Herein, GCPII and GCPIII are collectively referred to as GCP.
GCP is a highly expressed enzyme found on the surface of a number of cell types, including astrocytes and non-myelinating Schwann cells Inhibition of GCP-catalyzed NAAG hydrolysis is a promising therapy for the treatment of a number of neurodegenerative diseases. Indeed, genetic and pharmacological inhibition of GCP is neuroprotective in a variety of cell and animal disease models, and several GCP inhibitors are being investigated as therapeutics for the treatment of peripheral neuropathy and neuropathic pain.
Clinical development of a therapeutic agent (e.g., a GCP inhibitor) often use pharmacodynamic (PD) marker assays to determine what doses of the agent are required to elicit a pharmacologic response. Previously, NAAG levels in cerebrospinal fluid was the most common PD marker for monitoring GCP inhibition. However, collection of cerebrospinal fluid requires considerable skill and it is uncomfortable for patients. Also, NAAG measurement requires complicated laboratory techniques, such as HPLC or LC-MS/MS, and NAAG levels are only an indirect indicator of GCP inhibition.
Direct quantification of GCP enzyme activity would provide a more straightforward and accurate measurement of GCP inhibition. However, direct GCP activity measurement was thought to be unfeasible for use in PD marker assays in the clinic because GCP was believed to be predominantly expressed in the nervous system, prostate, intestinal tract, and kidney, tissues that are not easily accessible for collection from patients (See, e.g., Slusher et al., J. Biol. Chem. 265:21297-21301 (1990)). Previous attempts to quantify GCP activity in more accessible tissues, such as the skin, have been unsuccessful (See, e.g., Rovenska et al., Prostate 68:171-182 (2008)). Thus, there is a need for new methods for quantitating GCP activity in easily obtainable biological samples, such as skin biopsies.